One important area of application of imaging that has not been fully exploited, due to limitation of existing technology, is the ability to monitor the expression of specific cellular proteins in vivo. Unlike anatomical imaging, molecular imaging of specific cellular proteins would display the biochemical abnormalities underlying disease rather than the structural consequences of abnormalities. If fully developed, it could offer the opportunity to monitor the progress of clinical treatments by imaging the expression of specific marker proteins, and may even form an important platform to enable the testing and development of new therapeutic paradigms. The focus of this grant proposal is to harness the power of RNA-based sensors that will enable sensitive detection of specific molecular signatures in living cells. The RNA sensor we engineered controls the expression of a reporter gene by polyA signal-mediated cleavage. Mammalian polyA signals are exclusively located at the 3'-untranslated region (UTR). When a new polyA site is artificially created at 5' UTR, where they are never localized in normal transcriptional units, extremely efficient cleavage of that polyA signal leads to destruction of the mRNA and therefore loss of reporter gene expression. Binding of a target protein to the engineered polyA signal efficiently blocks the cleavage, resulting in preservation of the intact mRNA, thus enabling reporter expression. In turn, we have shown that the reporter signal from such a sensor exhibited extremely low leaky expression in live human cells, and upon the detection of a specific protein, the signal was effectively induced above one hundred folds. This is two orders of magnitude higher than has been previously achieved in live human cells, giving a dynamic range that would allow unprecedented applications in a variety of experimental settings. The overall objective is to create a general molecular sensor platform based on the modulation of polyA cleavage that could utilize current or next generation reporters and aptamers for the purpose of imaging a variety of specific molecules in live cells. Moreover, the established molecular sensor platform will provide a foundation for expanding the spectrum of molecular signatures that the polyA sensor can detect in vivo.